BRCA1 was identified as a gene frequently mutated in familial breast and ovarian cancer. Surprisingly, however, the frequency of BRCA1 mutation in sporadic cancer is very low. We have shown that while BRCA1 dysfunction may be more broadly involved in breast tumorigenesis than previously thought, and that defects in the nuclear translocation of BRCA1 protein may be a common determinant of breast cancer. Thus, we propose to investigate the molecular basis by which BRCA1 is translocated to the nucleus in normal cells and how the malfunction of this mechanism in breast epithelial cells leads to cancer. Our specific Aims are: (1) To confirm that a defective transportation process is a common cause of BRCA1 mislocation in advanced breast tumor cells, by examining the localization of exogenous, epitope- tagged, wild-type BRCA1 in normal cells and breast tumor lines by immunostaining and cell fractionation. We will also determine the nuclear localization signal (NLS) motif of BRCA1 by assessing the ability of mutants of the putative BRCA1 NLS motifs to translocate correctly to the nucleus. (2) To identify specific BRCA1 interacting proteins that are required for its correct localization, using the complementary methods of yeast two-hybrid screening and co- immunoprecipitation. (3) To test whether expression of proteins identified in aim 2 that are mutated in advanced breast cancer cell lines can rescue the transport of endogenous or ectopically expressed BRCA1 protein into the nucleus of breast tumor cells. (4) To assess the role of the identified genes in breast cancer development and progression. To do this, we will develop both immunostaining procedures and PCR-based SSCP assays to screen both premalignant breast lesions and primary invasive tumor specimens from the tumor Bank and Data Network Core, so as to establish any potential correlation between tumor development, prognosis, and mutation of the BRCA1 nuclear-transport protein. We have already made significant progress with the initial aims of this project. Results obtained from this investigation should provide considerable insight into the mechanism of BRCA1 transport and, in so doing, may identify other genes important in breast cancer. By combining both novel basic research and clinical correlation, we hope to provide information that will lead to better screening, prognostic assessment, and treatment of the most common fatal disease of women in developed countries.